A novel microseeding method for the crystallization of membrane proteins in lipidic cubic phase.
A novel microseeding method for the crystallization of membrane proteins in lipidic cubic phase.
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DOI：
10.1107/s2053230x16004118
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发表时间：
2016-04
期刊：
影响因子：
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通讯作者：
S. Kolek;Bastian Bräuning;P. Stewart
中科院分区：
文献类型：
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作者：
S. Kolek;Bastian Bräuning;P. Stewart
随机微晶种基质筛选（rMMS）是将晶种添加到随机结晶筛选中，这是可溶性蛋白质结晶的一项重要突破，它增加了可用于优化的结晶命中数。这大大增加了典型的结构生物学实验室每年产生的可溶性蛋白质结构的数量。受到这一成功的启发，rMMS已经适应于膜蛋白的结晶，通过放大LCP结晶条件而不改变对结晶至关重要的物理和化学参数来制备LCP种子原料。晶种直接在LCP中生长，并且与常规的rMMS一样，晶种实验与添加剂实验相结合。新方法用于细菌完整膜蛋白OmpF，发现它将结晶命中的数量增加了几乎一个数量级：在没有微接种的情况下发现了一个新的命中，而使用LCP-rMMS发现了八个新的命中。预计这种新方法将导致更好的膜蛋白晶体衍射。产生种子梯度的方法，它允许的LCP种子股票被稀释，并在每个LCP丸的晶体数量减少，如果需要优化，也证明。
Random microseed matrix screening (rMMS), in which seed crystals are added to random crystallization screens, is an important breakthrough in soluble protein crystallization that increases the number of crystallization hits that are available for optimization. This greatly increases the number of soluble protein structures generated every year by typical structural biology laboratories. Inspired by this success, rMMS has been adapted to the crystallization of membrane proteins, making LCP seed stock by scaling up LCP crystallization conditions without changing the physical and chemical parameters that are critical for crystallization. Seed crystals are grown directly in LCP and, as with conventional rMMS, a seeding experiment is combined with an additive experiment. The new method was used with the bacterial integral membrane protein OmpF, and it was found that it increased the number of crystallization hits by almost an order of magnitude: without microseeding one new hit was found, whereas with LCP-rMMS eight new hits were found. It is anticipated that this new method will lead to better diffracting crystals of membrane proteins. A method of generating seed gradients, which allows the LCP seed stock to be diluted and the number of crystals in each LCP bolus to be reduced, if required for optimization, is also demonstrated.